Many molecular targets suitable for antibody-mediated disease therapy have been validated with the use of non-human antibody reagents, and this process will continue for many of the new therapeutic targets which are expected to emerge from the human genome in the coming years. As a target becomes validated for therapy, the antibodies, typically murine, used to validate the target become leads for the development of biologic drugs. However, for many therapeutic applications the efficacy and safety of non-human antibodies are compromised because of their immunogenicity in patients. Thus, before such antibodies can be approved for therapeutic use they must be replaced with human counterparts having equivalent bioactivity, or they must be “humanized” in some way to eliminate or minimize immunogenicity in humans.
Established methods for the isolation of antigen-specific human antibodies include the screening of hybridomas from mice that are transgenic for the human immunoglobulin loci (e.g., Jakobavits, 1998, Adv Drug Deliv Rev. 31:33-42), and in vitro methods in which recombinant libraries of human antibody fragments displayed on and encoded in filamentous bacteriophage (e.g., McCafferty et al., 1990, Nature 348:552-554), yeast cells (e.g., Boder and Wittrup, 1997, Nat Biotechnol 15:553-557), and ribosomes (e.g., Hanes and Pluckthun, 1997, Proc Natl Acad Sci USA 94:4937-4942) are panned against immobilized antigen. These methods have yielded many useful human antibodies. However, for many non-human antibodies with desirable therapeutic properties, human antibodies with equivalent bioactivities have not been isolated using these methods.
Mice transgenic for human immunoglobulin loci generally do not express the full complement of human diversity, and affinity maturation is less efficient. Thus, the success rates for desired affinities and specificities tend to be lower than with conventional mice. The principal limitations of the display technologies stem from biased expression of antibody repertoires, and the specificity and affinity limitations of naïve repertoires. Antigen-binding antibodies from naïve libraries typically require additional affinity maturation, which with current in vitro methods is an arduous and uncertain process and moreover, may introduce immunogenic epitopes into the antibody.
The most widely used methods for minimizing the immunogenicity of non-human antibodies while retaining as much of the original specificity and affinity as possible involve grafting the CDRs of the non-human antibody onto human frameworks typically selected for their structural homology to the non-human framework (Jones et al., 1986, Nature 321:522-5; U.S. Pat. No. 5,225,539). Originally these methods resulted in drastic losses of affinity. However, it was then shown that some of the affinity could be recovered by restoring the non-human residues at key positions in the framework that are required to maintain the canonical structures of the non-human CDRs 1 and 2 (Bajorath et al., 1995, J Biol Chem 270:22081-4; Martin et al., 1991, Methods Enzymol. 203:121-53; Al-Lazikani, 1997, J Mol Biol 273:927-48). Recovering the native conformations of CDR3s is a much more uncertain enterprise because their structures are more variable. Determining which non-human residues to restore to recover functional CDR3 conformation is thus largely a matter of modeling where possible combined with trial and error. As a result, in many cases the full affinity of the original non-human antibody is not recovered. Exemplary methods for humanization of antibodies by CDR grafting are disclosed, for example, in U.S. Pat. No. 6,180,370.
To mitigate the shortcomings of the traditional CDR-grafting approaches, various hybrid selection approaches have been tried, in which portions of the non-human antibody have been combined with libraries of complementary human antibody sequences in successive rounds of selection for antigen binding, in the course of which most of the non-human sequences are gradually replaced with human sequences. These approaches have generally not fared better than CDR-grafting, however. For example, in the chain-shuffling technique (Marks, et al., 1992, Biotechnology 10:779-83) one chain of the non-human antibody is combined with a naïve human repertoire of the other chain on the rationale that the affinity of the non-human chain will be sufficient to constrain the selection of a human partner to the same epitope on the antigen. Selected human partners are then used to guide selection of human counterparts for the remaining non-human chains.
Other methodologies include chain replacement techniques where the non-human CDR3s were retained and only the remainder of the V-regions, including the frameworks and CDRs 1 and 2, were individually replaced in steps performed sequentially (e.g., U.S. Patent Application No. 20030166871; Rader, et al., Proc Natl Acad Sci USA 95:8910-15, 1998; Steinberger, et al., J. Biol. Chem. 275:36073-36078, 2000; Rader, et al., J. Biol. Chem. 275:13668-13676, 2000). However, this strategy still has the drawback that selectable human V-regions must be compatible not just with the non-human CDR3 but with the non-human companion V-region as well. This inter-species compatibility imposes a high demand for structural homology in the selected human V-regions, such that those most homologous to the non-human V-regions are generally selected. In this regard, the result is quite similar to that of the CDR grafting approach, except that CDRs 1 and 2 are initially human in the selected V-regions.
Existing methods for the isolation of human antibodies with required bioactivities for therapeutic use, or for “humanizing” non-human antibodies for therapeutic use thus have many limitations, as noted above. Primarily, these limitations relate to the retention or inclusion of nonhuman sequences in order to maintain binding affinity. Thus, there is a need for efficient humanization methods that minimize nonhuman sequences and thereby minimize introduction of immunogenic epitopes. The current invention addresses this need.